Sterols are steroid alcohols of vegetable and animal origin. Ergosterol is the principal membrane sterol of fungi. It is structurally similar to its animal counterpart, cholesterol, and its higher plant counterparts, stigmasterol and sitosterol. Though the biosynthesis of ergosterol in fungi involves steps distinct from the other sterols, the pathways in different organisms share several common steps. The lanosterol 14.alpha.-demethylation steps in cholesterol and ergosterol formation in animals and fungi, as well as the obtusifoliol 14.alpha.-demethylation in stigmasterol and sitosterol biosynthesis in plants, both lead to the formation of a double bond between carbons 14 and 15 of the sterol ring. This double bond is then reduced by sterol .DELTA.14 reductase activity. The enzyme is located in the microsomal fraction in pig liver, yeast and Zea mays, and requires NADPH as an electron donor (Marcireau, C., et al., Curr. Genet. 22: 267-272 (1992)).
Genetic studies of ergosterol biosynthesis mainly have been carried out in Saccharomyces cerevisiae (Paltauf, F., et al., in Jones, E. W., et al., eds., The Molecular and Cellular Biology of the Yeast Saccharomyces, Gene Expression, Cold Spring Harbor Laboratory Press, 1992, pages 434-437). In yeast, ergosterol affects membrane fluidity and permeability and plays an essential role in the yeast cell cycle.
A number of mutations in the yeast ergosterol biosynthetic pathway have been isolated either by reverse genetic approaches or by selection for mutations producing polyene resistance, and many of the genes have been identified. Toward the end of the pathway, sterol .DELTA.14 reductase, .DELTA.8-.DELTA.7 isomerase, and C-24(28) reductase catalyze steps in the conversion of lanosterol to ergosterol. After ignosterol is reduced by sterol .DELTA.14 reductase, which eliminates a double bond in the D ring of the molecule, the sterol is demethylated and rearranged to fecosterol, which is then isomerized by sterol .DELTA.8-.DELTA.7 isomerase. The sterol is then desaturated in two positions and its side chain is reduced by C-24(28) reductase. Some of the genes encoding the enzymes have been identified and named as follows (Paultauf, et al., cited above, Lorenz, T., and Parks, L. W., DNA and Cell Biol. 11: 685-692 (1992), and Example 1 below):
______________________________________ Enzyme Gene ______________________________________ .DELTA.14 reductase ERG24 C-24 methyl transferase ERG6 .DELTA.8-.DELTA.7 isomerase ERG2 C-24(28) reductase ERG4 ______________________________________
Based on the accumulation of intermediates following fungicide treatment, morpholine fungicidal compounds such as tridemorph and fenpropimorph have been reported to be inhibitors of sterol 14 reductase and sterol .DELTA.8-.DELTA.7 isomerase (Baloch, R. and Mercer, I., Phytochemistry 26: 663-668 (1987)). However, it recently has been found that the sterol .DELTA.8-.DELTA.7 isomerase gene is not essential for viability in S. cerevisiae (Ashman, W. H., et al., Lipids 26: 628-632 (1991)), suggesting that the killing effect of morpholine fungicides may be primarily the result of sterol .DELTA.14 reductase inhibition.
It has also been shown that the C-24 methyl transferase gene (ERG6) is not essential for viability in S. cerevisiae (Gaber, R. F., et al., Mol. Cell. Biol. 9: 3447-3456 (1989)). Mutant cells exhibit normal vegetative growth, but they differ from the wildtype in a number of respects, including drug supersensitivity, presumably due to alterations in membrane function (ibid.). Drug super-sensitivity has been observed in other yeast mutants, including one denoted YGL022 which encodes a putative transport protein (Chen, W., et al., Yeast 7: 305-308 (1991)).